Project description:We performed all stimulatory experiments using THP1 cell line as a representative of primary human monocytes to show fundamental role of the cfDNA in healthy organisms. The experiments were conducted in duplicates using plasma containing cfDNA (NP) and the reference one with cfDNA removed by DNase (TP) to recognize unequivocally the effect of plasma cfDNA on transcriptome and proteome of monocytes. We used native human plasma samples obtained from healthy volunteers with no animal serum addition to cultivation medium in order to avoid the presence of uncharacterized animal cfDNA in the experiments.
Project description:BACKGROUND:Plasma-based cell-free DNA is an attractive biospecimen for assessing somatic mutations due to minimally-invasive real-time sampling. However, next generation sequencing (NGS) of cell-free DNA (cfDNA) may not be appropriate for all patients with advanced prostate cancer (PC). METHODS:Blood was obtained from advanced PC patients for plasma-based sequencing. UW-OncoPlex, a ?2?Mb multi-gene NGS panel performed in the CLIA/CAP environment, was optimized for detecting cfDNA mutations. Tumor tissue and germline samples were sequenced for comparative analyses. Multivariate logistic regression was performed to determine the clinical characteristic associated with the successful detection of somatic cfDNA alterations (ie detection of at least one clearly somatic PC mutation). RESULTS:Plasma for cfDNA sequencing was obtained from 93 PC patients along with tumor tissue (N?=?67) and germline (N?=?93) controls. We included data from 76 patients (72 prostate adenocarcinoma; 4 variant histology PC) in the analysis. Somatic DNA aberrations were detected in 34 cfDNA samples from patients with prostate adenocarcinoma. High PSA level, high tumor volume, and castration-resistance were significantly associated with successful detection of somatic cfDNA alterations. Among samples with somatic mutations detected, the cfDNA assay detected 93/102 (91%) alterations found in tumor tissue, yielding a clustering-corrected sensitivity of 92% (95% confidence interval 88-97%). All germline pathogenic variants present in lymphocyte DNA were also detected in cfDNA (N?=?12). Somatic mutations from cfDNA were detected in 30/33 (93%) instances when PSA was >10?ng/mL. CONCLUSIONS:Disease burden, including a PSA >10?ng/mL, is strongly associated with detecting somatic mutations from cfDNA specimens.
Project description:BACKGROUND:Liquid biopsies using circulating tumor DNA (ctDNA) and cell-free DNA (cfDNA) have been developed for early cancer detection and patient monitoring. To investigate the clinical usefulness of ctDNA aberrations and cfDNA levels in patients with breast cancer (BC), we conducted a meta-analysis of 69 published studies on 5736 patients with BC. METHODS:The relevant publications were identified by searching PubMed and Embase databases. The effect sizes of outcome parameters were pooled using a random-effects model. RESULTS:The ctDNA mutation rates of TP53, PIK3CA, and ESR1 were approximately 38%, 27%, and 32%, respectively. High levels of cfDNA were associated with BCs rather than with healthy controls. However, these detection rates were not satisfactory for BC screening. Although the precise mechanisms have been unknown, high cfDNA levels were significantly associated with axillary lymph node metastasis (odds ratio [OR]?=?2.148, P?=?.030). The ctDNA mutations were significantly associated with cancer recurrence (OR?=?3.793, P?<?.001), short disease-free survival (univariate hazard ratio [HR]?=?5.180, P?=?.026; multivariate HR?=?3.605, P?=?.001), and progression-free survival (HR?=?1.311, P?=?.013) rates, and poor overall survival outcomes (HR?=?2.425, P?=?.007). CONCLUSION:This meta-analysis demonstrates that ctDNA mutation status predicts disease recurrence and unfavorable survival outcomes, while cfDNA levels can be predictive of axillary lymph node metastasis in patients with BC.
Project description:<h4>Background</h4>The comparison between relatively intact nanoscale extracellular vesicle-derived DNA (nEV-DNA) and fragmented circulating cell-free DNA (cfDNA) in mutation detection among patients with non-small-cell lung cancer (NSCLC) has not been carried out yet, and thus deserves investigation.<h4>Patients and methods</h4>Both nEV-DNA and cfDNA was obtained from 377 NSCLC patients with known EGFR mutation status and 69 controls. The respective EGFRE19del/T790M/L858R mutation status was interrogated with amplification-refractory-mutation-system-based PCR assays (ARMS-PCR).<h4>Results</h4>Neither nEV-DNA nor cfDNA levels show a strong correlation with tumor volumes. There is no correlation between cfDNA and nEV-DNA levels either. The detection sensitivity of nEV-DNA and cfDNA using ARMS-PCR in early-stage NSCLC was 25.7% and 14.2%, respectively, with 96.6% and 91.7% specificity, respectively. In late-stage NSCLC, both nEV-DNA and cfDNA show ∼80% sensitivity and over 95% specificity.<h4>Conclusions</h4>nEV-DNA is superior to cfDNA for mutation detection in early-stage NSCLC using ARMS-PCR. However, the advantages vanish in late-stage NSCLC.
Project description:Patients with Lynch syndrome carry germline mutations in single alleles of genes encoding the mismatch repair (MMR) proteins MLH1, MSH2, MSH6, and PMS2; when the second allele becomes mutated, cancer can develop. Increased screening for Lynch syndrome has identified patients with tumors that have deficiency in MMR, but no germline mutations in genes encoding MMR proteins. We investigated whether tumors with deficient MMR had acquired somatic mutations in patients without germline mutations in MMR genes using next-generation sequencing.We analyzed blood and tumor samples from 32 patients with colorectal or endometrial cancer who participated in Lynch syndrome screening studies in Ohio and were found to have tumors with MMR deficiency (based on microsatellite instability and/or absence of MMR proteins in immunohistochemical analysis, without hypermethylation of MLH1), but no germline mutations in MMR genes. Tumor DNA was sequenced for MLH1, MSH2, MSH6, PMS2, EPCAM, POLE, and POLD1 with ColoSeq and mutation frequencies were established.Twenty-two of 32 patients (69%) were found to have 2 somatic (tumor) mutations in MMR genes encoding proteins that were lost from tumor samples, based on immunohistochemistry. Of the 10 remaining tumors 3 had one somatic mutation in a MMR gene, with possible loss of heterozygosity that could lead to MMR deficiency, 6 were found to be false-positive results (19%), and 1 had only one mutation in a MMR gene and remained unexplained. All of the tumors found to have somatic MMR mutations were of the hypermutated phenotype (>12 mutations/megabase); 6 had mutation frequencies >200/megabase, and 5 of these had somatic mutations in POLE, which encodes a DNA polymerase.Some patients are found to have tumors with MMR defects during screening for Lynch syndrome, yet have no identifiable germline mutations in MMR genes. We found that almost 70% of these patients acquire somatic mutations in MMR genes, leading to a hypermutated phenotype of tumor cells. Patients with colon or endometrial cancers with MMR deficiency not explained by germline mutations might undergo analysis for tumor mutations in MMR genes to guide future surveillance guidelines.
Project description:Circulating cell-free DNA from blood plasma of cancer patients can be used to non-invasively interrogate somatic tumor alterations. Here we develop MSK-ACCESS (Memorial Sloan Kettering - Analysis of Circulating cfDNA to Examine Somatic Status), an NGS assay for detection of very low frequency somatic alterations in 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 99% for a priori mutation profiling. To evaluate the performance of MSK-ACCESS, we report results from 681 prospective blood samples that underwent clinical analysis to guide patient management. Somatic alterations are detected in 73% of the samples, 56% of which have clinically actionable alterations. The utilization of matched normal sequencing allows retention of somatic alterations while removing over 10,000 germline and clonal hematopoiesis variants. Our experience illustrates the importance of analyzing matched normal samples when interpreting cfDNA results and highlights the importance of cfDNA as a genomic profiling source for cancer patients.
Project description:To better understand the etiology of inflammatory breast cancer (IBC) and identify potential therapies, we studied genomic alterations in IBC patients. Targeted, next-generation sequencing (NGS) was performed on cell-free DNA (cfDNA) (<i>n</i> = 33) and paired DNA from tumor tissues (<i>n</i> = 29) from 32 IBC patients. We confirmed complementarity between cfDNA and tumor tissue genetic profiles. We found a high incidence of germline variants in IBC patients that could be associated with an increased risk of developing the disease. Furthermore, 31% of IBC patients showed deficiencies in the homologous recombination repair (HRR) pathway (BRCA1, BRCA2, PALB2, RAD51C, ATM, BARD1) making them sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors. We also characterized the tumor-infiltrating lymphocytes (TILs) in tumor tissue biopsies by studying several markers (CD4, CD8, FoxP3, CD20, PD-1, and PD-L1) through immunohistochemistry (IHC) staining. In 7 of 24 (29%) patients, tumor biopsies were positive for PD-L1 and PD-1 expression on TILs, making them sensitive to PD-1/PD-L1 blocking therapies. Our results provide a rationale for considering PARP inhibitors and PD-1/PDL1 blocking immunotherapy in qualifying IBC patients.
Project description:The use of blood-circulating cell-free DNA (cfDNA) as 'liquid-biopsy' is explored worldwide, with hopes for its potential in providing prognostic or predictive information in cancer treatment. In exploring cfDNA, valuable repositories are biobanks containing material collected over time, however these retrospective cohorts have restrictive resources. In this study, we aimed to detect tumor-specific mutations in only minute amounts of serum-derived cfDNA by using a targeted next generation sequencing (NGS) approach. In a retrospective cohort of ten metastatic breast cancer patients, we profiled DNA from primary tumor tissue (frozen), tumor-adjacent normal tissue (formalin-fixed paraffin embedded), and three consecutive serum samples (frozen). Our presented workflow includes comparisons with matched normal DNA or in silico reference DNA to discriminate germline from somatic variants, validation of variants through the detection in at least two DNA samples of an individual, and the use of public databases on variants. By our workflow, we were able to detect a total of four variants traceable as circulating tumor DNA (ctDNA) in the sera of three of the ten patients.
Project description:Genomic analysis of tumor tissue is the standard technique for identifying DNA alterations in malignancies. Genomic analysis of circulating tumor cell-free DNA (cfDNA) represents a relatively non-invasive method of assessing genomic alterations using peripheral blood. We compared the concordance of genomic alterations between cfDNA and tissue biopsies in this retrospective study. Twenty-eight patients with advanced solid tumors with paired next-generation sequencing tissue and cfDNA biopsies were identified. Sixty-five genes were common to both assays. Concordance was defined as the presence or absence of the identical genomic alteration(s) in a single gene on both molecular platforms. Including all aberrations, the average number of alterations per patient for tissue and cfDNA analysis was 4.82 and 2.96, respectively. When eliminating alterations not detectable in the cfDNA assay, mean number of alterations for tissue and cfDNA was 3.21 and 2.96, respectively. Overall, concordance was 91.9-93.9%. However, the concordance rate decreased to 11.8-17.1% when considering only genes with reported genomic alterations in either assay. Over 50% of mutations detected in either technique were not detected using the other biopsy technique, indicating a potential complementary role of each assay. Across 5 genes (TP53, EGFR, KRAS, APC, CDKN2A), sensitivity and specificity were 59.1% and 94.8%, respectively. Potential explanations for the lack of concordance include differences in assay platform, spatial and temporal factors, tumor heterogeneity, interval treatment, subclones, and potential germline DNA contamination. These results highlight the importance of prospective studies to evaluate concordance of genomic findings between distinct platforms that ultimately may inform treatment decisions.